Lipid Composition Matters: POPE Enrichment Modulates Curvature and Stress Redistribution in Gram-Negative Outer Membrane Models

When studying biological membranes, one important aspect to consider is lipid composition and asymmetry, since both play critical roles in determining membrane structure, mechanical properties, and biological function. Gram-negative bacteria provide a notable example, with an asymmetric outer membrane (OM) composed mainly of lipopolysaccharides (LPS) in the outer leaflet and phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) in the inner leaflet. The maintenance of membrane asymmetry is regulated by transport systems, e.g., the Mla pathway, which removes phospholipids from the outer leaflet, preserving membrane integrity. Mutations in the Mla pathway destabilize the membrane and increase susceptibility to antimicrobial compounds. In addition, PE enrichment has been proposed as a step toward outer membrane vesicle (OMV) formation, contributing to bacterial resistance. Despite the importance of PE enrichment, the molecular details remain unclear. The present study investigated the consequences of enriching OM models with PE. To do this, MD simulations of asymmetric membranes were combined with SAXS, DLS, and zeta-potential measurements on membrane mimetics with increasing PE amounts (0%, 25%, 50%, 75%, and 100% PE). The results showed that PE enrichment drives a gradual mechanical transition. At 0% PE, the membrane is mechanically tense and curvature frustrated, but with PE enrichment the system becomes more stress homogeneous and relaxed through curvature formation. At the molecular level, PE and LPS mix favorably, with negative excess area. A reduction in unevenness of lateral stress and its redistribution within membranes was also observed. The simulations further showed that LPS forms clusters, driving local saddle-like curvature, linking local composition heterogeneity to membrane remodeling. Moving to the mesoscale, SAXS reveals a structural shift: pure LPS scatters like flexible polymer-like aggregates, whereas PE enriched systems form ordered lamellar structures. The hydrodynamic radius grows from 50 to 500 nm, and the surface charge becomes less negative with PE enrichment, suggesting that electrostatics may control assembly size and stability. Taken together, these results support a model where PE accumulation remodels the OM by modulating stress distribution and promoting curvature as a relaxation mechanism; providing a direct molecular link between lipid composition, membrane mechanics, and early step of OMV formation.